NG25

NG25, a TAK1 inhibitor, inhibited the activation of IKK by TLR7 and TLR9 agonists and prevented the secretion of type 1 IFNs induced by these ligands in Gen2.2 cells.

TAK1 inhibitor NG25 enhances doxorubicin-mediated apoptosis in breast cancer cells.[Pubmed:27599572]

Sci Rep. 2016 Sep 7;6:32737.

Doxorubicin (Dox, Adriamycin) has been widely used in breast cancer treatment. But its severe cardio-toxic side effects limited the clinical use. Dox treatment can induce DNA damage and other accompanying effects in cancer cells, and subsequently activates nuclear factor kappaB (NF-kappaB) pathway which has a strong pro-survival role in different types of malignancy. We hypothesize that blocking NF-kappaB pathway may sensitize breast cancer cells to Dox chemotherapy. TGFbeta-activated kinase-1 (TAK1) is a key intracellular molecule participating in genotoxic stresses-induced NF-kappaB activation. Targeting TAK1 as a strategy to enhance cancer treatment efficacy has been studied in several malignancies. We showed that NG25, a synthesized TAK1 inhibitor, greatly enhanced Dox treatment efficacy in a panel of breast cancer cell lines. In this pre-clinical study, we found that NG25 partially blocked Dox-induced p38 phosphorylation and IkappaBalpha degradation and enhanced Dox-induced cytotoxic effects and apoptosis in all breast cancer cell lines tested. Taken together, we provided clear evidence that NG25 sensitizes the breast cancer cells to Dox treatment in vitro. This combination may be an effective and feasible therapeutic option maximizing Dox efficacy and meanwhile minimizing Dox side effects in treating breast cancer.

Discovery of type II inhibitors of TGFbeta-activated kinase 1 (TAK1) and mitogen-activated protein kinase kinase kinase kinase 2 (MAP4K2).[Pubmed:25075558]

J Med Chem. 2015 Jan 8;58(1):183-96.

We developed a pharmacophore model for type II inhibitors that was used to guide the construction of a library of kinase inhibitors. Kinome-wide selectivity profiling of the library resulted in the identification of a series of 4-substituted 1H-pyrrolo[2,3-b]pyridines that exhibited potent inhibitory activity against two mitogen-activated protein kinases (MAPKs), TAK1 (MAP3K7) and MAP4K2, as well as pharmacologically well interrogated kinases such as p38alpha (MAPK14) and ABL. Further investigation of the structure-activity relationship (SAR) resulted in the identification of potent dual TAK1 and MAP4K2 inhibitors such as 1 (NG25) and 2 as well as MAP4K2 selective inhibitors such as 16 and 17. Some of these inhibitors possess good pharmacokinetic properties that will enable their use in pharmacological studies in vivo. A 2.4 A cocrystal structure of TAK1 in complex with 1 confirms that the activation loop of TAK1 assumes the DFG-out conformation characteristic of type II inhibitors.

The IkappaB kinase family phosphorylates the Parkinson's disease kinase LRRK2 at Ser935 and Ser910 during Toll-like receptor signaling.[Pubmed:22723946]

PLoS One. 2012;7(6):e39132.

Mutations in leucine-rich repeat kinase 2 (LRRK2) are strongly associated with late-onset autosomal dominant Parkinson's disease. LRRK2 is highly expressed in immune cells and recent work points towards a link between LRRK2 and innate immunity. Here we demonstrate that stimulation of the Toll-Like Receptor (TLR) pathway by MyD88-dependent agonists in bone marrow-derived macrophages (BMDMs) or RAW264.7 macrophages induces marked phosphorylation of LRRK2 at Ser910 and Ser935, the phosphorylation sites that regulate the binding of 14-3-3 to LRRK2. Phosphorylation of these residues is prevented by knock-out of MyD88 in BMDMs, but not the alternative TLR adaptor protein TRIF. Utilising both pharmacological inhibitors, including a new TAK1 inhibitor, NG25, and genetic models, we provide evidence that both the canonical (IKKalpha and IKKbeta) and IKK-related (IKKepsilon and TBK1) kinases mediate TLR agonist induced phosphorylation of LRRK2 in vivo. Moreover, all four IKK members directly phosphorylate LRRK2 at Ser910 and Ser935 in vitro. Consistent with previous work describing Ser910 and Ser935 as pharmacodynamic biomarkers of LRRK2 activity, we find that the TLR independent basal phosphorylation of LRRK2 at Ser910 and Ser935 is abolished following treatment of macrophages with LRRK2 kinase inhibitors. However, the increased phosphorylation of Ser910 and Ser935 induced by activation of the MyD88 pathway is insensitive to LRRK2 kinase inhibitors. Finally, employing LRRK2-deficient BMDMs, we present data indicating that LRRK2 does not play a major role in regulating the secretion of inflammatory cytokines induced by activation of the MyD88 pathway. Our findings provide the first direct link between LRRK2 and the IKKs that mediate many immune responses. Further work is required to uncover the physiological roles that phosphorylation of LRRK2 by IKKs play in controlling macrophage biology and to determine how phosphorylation of LRRK2 by IKKs impacts upon the use of Ser910 and Ser935 as pharmacodynamic biomarkers.

Essential role for IKKbeta in production of type 1 interferons by plasmacytoid dendritic cells.[Pubmed:22511786]

J Biol Chem. 2012 Jun 1;287(23):19216-28.

Plasmacytoid dendritic cells (pDCs) are characterized by their ability to produce high levels of type 1 interferons in response to ligands that activate TLR7 and TLR9, but the signaling pathways required for IFN production are incompletely understood. Here we exploit the human pDC cell line Gen2.2 and improved pharmacological inhibitors of protein kinases to address this issue. We demonstrate that ligands that activate TLR7 and TLR9 require the TAK1-IKKbeta signaling pathway to induce the production of IFNbeta via a pathway that is independent of the degradation of IkappaBalpha. We also show that IKKbeta activity, as well as the subsequent IFNbeta-stimulated activation of the JAK-STAT1/2 signaling pathway, are essential for the production of IFNalpha by TLR9 ligands. We further show that TLR7 ligands CL097 and R848 fail to produce significant amounts of IFNalpha because the activation of IKKbeta is not sustained for a sufficient length of time. The TLR7/9-stimulated production of type 1 IFNs is inhibited by much lower concentrations of IKKbeta inhibitors than those needed to suppress the production of NFkappaB-dependent proinflammatory cytokines, such as IL-6, suggesting that drugs that inhibit IKKbeta may have a potential for the treatment of forms of lupus that are driven by self-RNA and self-DNA-induced activation of TLR7 and TLR9, respectively.